Silk biomaterials and methods of use thereof

ABSTRACT

The present invention provides an all-aqueous process and composition for production of silk biomaterials, e.g., fibers, films, foams and mats. In the process, at least one biocompatible polymer, such as poly(ethylene oxide) (PEO) (a well-documented biocompatible material), was blended with the silk protein prior to processing e.g., electrospinning. We discovered that this step avoids problems associated with conformational transitions of fibroin during solubilization and reprocessing from aqueous solution which lead to embrittled materials. Moreover, the process avoids the use of organic solvents that can pose problems when the processed biomaterials are exposed to cells in vitro or in vivo.

GOVERNMENT SUPPORT

The subject matter of this application was made with support from theUnited States Government, National Institutes of Health (NIH) grant R01DE13405-01A1, National Science Foundation grant DMR0090384, and UnitedStates Air Force grant F49620-01-C-0064.

FIELD OF THE INVENTION

The present invention relates generally to silk biomaterials, e.g.,fibers, films, foams and mats, and use of those materials in tissueengineered constructs.

BACKGROUND OF THE INVENTION

Electrospinning for the formation of fine fibers has been activelyexplored recently for applications such as high performance filters [1,2] and biomaterial scaffolds for cell growth, vascular grafts, wounddressings or tissue engineering [2-4]. Fibers with nanoscale diameterprovide benefits due to their high surface area. In this electrostatictechnique, a strong electric field is generated between a polymersolution contained in a glass syringe with a capillary tip and ametallic collection screen. When the voltage reaches a critical value,the charge overcomes the surface tension of the deformed drop ofsuspended polymer solution formed on the tip of the syringe, and a jetis produced. The electrically charged jet undergoes a series ofelectrically induced bending instabilities during passage to thecollection screen that results in stretching [5-7]. This stretchingprocess is accompanied by the rapid evaporation of the solvent andresults in a reduction in the diameter of the jet [8-12]. The dry fibersaccumulated on the surface of the collection screen form a non-wovenmesh of nanometer to micrometer diameter fibers even when operating withaqueous solutions at ambient temperature and pressure. Theelectrospinning process can be adjusted to control fiber diameter byvarying the charge density and polymer solution concentration, while theduration of electrospinning controls the thickness of the deposited mesh[8-13].

Protein fiber spinning in nature, such as for silkworm and spider silks,is based on the formation of concentrated solutions of metastablelyotropic phases that are then forced through small spinnerets into air[14]. The fiber diameters produced in these natural spinning processesrange from tens of microns in the case of silkworm silk to microns tosubmicron in the case of spider silks [14]. The production of fibersfrom protein solutions has typically relied upon the use of wet or dryspinning processes [15, 16]. Electrospinning offers an alternativeapproach to protein fiber formation that can potentially generate veryfine fibers. This would be a useful feature based on the potential roleof these types of fibers in some applications such as biomaterials andtissue engineering [17]. Electrospinning has been utilized to generatenanometer diameter fibers from recombinant elastin protein [17] andsilk-like protein [18-20]. Zarkoob et al. [21] have also reported thatsilkworm silk from Bombyx mori cocoons and spider dragline silk fromNephila clavipes silk can be electrospun into nanometer diameter fibersif first solubilized in the organic solvent hexafluoro-2-propanol (REP).

Silk is a well described natural fiber produced by the silkworm, Bombyxmori, which has been used traditionally in the form of threads intextiles for thousands of years. This silk contains a fibrous proteintermed fibroin (both heavy and light chains) that form the thread core,and glue-like proteins termed sericin that surround the fibroin fibersto cement them together. The fibroin is a highly insoluble proteincontaining up to 90% of the amino acids glycine, alanine and serineleading to pleated sheet formation in the fibers [22].

The unique mechanical properties of reprocessed silk such as fibroin andits biocompatibility make the silk fibers especially attractive for usein biotechnological materials and medical applications [14, 23].

Electrospinning silk fibers for biomedical applications is a complicatedprocess, especially due to problems encountered with conformationaltransitions of silkworm fibroin during solubilization and reprocessingfrom aqueous solution to generate new fibers and films. The problem withconformation transition is due to the formation of β-sheets which resultin embrittled materials. Additionally, organic solvents typically usedin silk electrospinning, as well as foam, film or mesh formation, posebiocompatibility problems when the processed materials are exposed tocells in vitro or in vivo.

Silk blends have been extensively studied with respect to filmformation. Blends with polyacrylamide [26], sodium alginate [27],cellulose [28, 35], chitosan [29, 36, 37], poly(vinyl alcohol) [30, 38,39], acrylic polymers [31], poly(ethylene glycol) (300 g/mol [40] or8,000 g/mol [41]) poly(ε-caprolactone-co-D,L-lactide) [42], andS-carboxymethyl keratin [43] have been studied to improve the mechanicalor thermal stability or membrane properties of silk films.

Unfortunately, none of these blends have proven successful in overcomingproblems associated with processing or reprocessing silk protein, e.g.,embrittlement, and, therefore, new methods, especially organic solventfree methods, are needed.

SUMMARY OF THE INVENTION

The present invention provides an all-aqueous process for production ofsilk biomaterials, e.g., fibers, films, foams and mats. In the process,at least one biocompatible polymer, such as poly(ethylene oxide) (PEO),was blended with the silk protein prior to processing e.g.,electrospinning. We discovered that this step avoids problems associatedwith conformational transitions of fibroin during solubilization andreprocessing from aqueous solution which lead to embrittled materials.Moreover, the process avoids the use of organic solvents that can poseproblems when the processed biomaterials are exposed to cells in vitroor in vivo.

In one embodiment, the biomaterial is a fiber. The fiber is produced bya process comprising the steps of (a) preparing an aqueous solution of asilk protein; (b) adding a biocompatible polymer to the aqueoussolution; and (c) electrospinning the solution. The process may furthercomprise step (d) of immersing the fiber into an alcohol/water solution.The alcohol is preferably methanol, ethanol, isopropyl alcohol(2-propanol) or n-butanol. Methanol is most preferred. Additionally, theprocess may further comprise step (e) of washing the fibroin fiber inwater.

The present invention also provides a fiber produced by the process.

In another embodiment, the biomaterial is a film. The film is produced,for example, by a process comprising the steps of (a) preparing anaqueous solution of a silk protein; (b) adding a biocompatible polymerto the aqueous solution; (c) drying the mixture; and (d) contacting themixture with an alcohol/water solution to crystallize the silk blendfilm. The process can optionally include step (e) of drawing ormono-axially stretching the resulting film to alter or enhance itsmechanical properties.

In the processes of the present invention, the aqueous solution of asilk protein is preferably in an aqueous salt solution (e.g., lithiumbromide or lithium thiocyanate) or a strong acid solution (e.g., formicacid, hydrochloric acid).

The silk protein suitable for use in the present invention is preferablyfibroin or related proteins (i.e., silks from spiders). The fibroin orrelated proteins are preferably obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk is obtained,for example, from Bombyx mori. Spider silk may be obtained from Nephilaclavipes. In the alternative, the silk protein suitable for use in thepresent invention can be obtained from a solution containing agenetically engineered silk, such as from bacteria, yeast, mammaliancells, transgenic animals or transgenic plants. See, for example, WO97/08315 and U.S. Pat. No. 5,245,012.

The present invention also provides a biomaterial comprising a silkprotein and a biocompatible polymer. The biomaterial may be a fiber,film, foam or a non-woven network of fibers (also referred to as a mat).The biomaterial may be used to facilitate tissue repair, ingrowth orregeneration as scaffold in a tissue engineered biocompatible polymerengineered construct, or to provide delivery of a protein or therapeuticagent.

As used herein, biocompatible means that the polymer is non-toxic,non-mutagenic, and elicits a minimal to moderate inflammatory reaction.Preferred biocompatible polymer for use in the present inventioninclude, for example, polyethylene oxide (PEO), polyethylene glycol(PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine,alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone,polylactic acid, polyglycolic acid, polyhydroxyalkanoates, dextrans, andpolyanhydrides. In accordance with the present invention, two or morebiocompatible polymers can be added to the aqueous solution.

The present invention further provides a composition comprising a silkprotein and a biocompatible polymer in water, wherein the composition isfree of solvents other than water. Preferably, the silk protein isfibroin and the biocompatible polymer is PEO. The composition is usefulin the methods of the present invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, the preferred methods and materials are described below. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference. In addition, thematerials, methods and examples are illustrative only and not intendedto be limiting. In case of conflict, the present specification,including definitions, controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain the objects, advantages,and principles of the invention.

FIG. 1 illustrates shear viscosities of silk/PEO blend solutions inwater.

FIG. 2 is a scanning electron micrograph of electrospun fibers (No. 6)and sericin extracted Bombyx mori silk fiber (500 magnification).

FIGS. 3A-3D are scanning electron micrographs of electrospun fibers (No.1): (a) an elecrospun fiber, (b) after methanol treatment, (c) afterdissolved in water at room temp and (d) after dissolved in water at36.5° C.

FIG. 4 is an ATR spectra of electrospun mat from silk/PEO blendsolutions (No. 6) (dotted line: after methanol/water (90/10 v/v)treatment).

FIGS. 5A-13B show percentage weight loss of silk and PEO blend films inwater at 37° C. (dotted line: calculated silk weight in films): (13A)silk/PEO blend and (13B) silk/PEG blend.

FIG. 6 shows DSC thermograms of silk, PEO and silk/PEO blend filmsbefore methanol treatment: (a) silk film; (b) silk/PEO (98/2) blend; (c)silk/PEO (90/10) blend; (d) silk/PEO (80/20) blend; (e) silk/PEO (70/30)blend; (f) silk/PEO (60/40) blend; and (g) PEO.

FIG. 7 show DSC thermograms of silk/PEO blend films after methanoltreatment: (a) silk film; (b) silk/PEO (98/2) blend; (c) silk/PEO(90/10) blend; (d) silk/PEO (80/20) blend; (e) silk/PEO (70/30) blend;and (f) silk/PEO (60/40) blend.

FIGS. 8A-8B show optical polarizing images of electrospun fibers (scalebar: 10 μM): (a) before heating at room temperature and (b) afterheating up 100° C. at a rate of 5° C./min.

FIG. 9 show differential scanning calorimeter (DSC) thermograms ofsilk/PEO electrospun fiber mats after methanol treatment: (a) PEOnon-extracted mat and (b) PEO extracted mat.

FIGS. 10A-10C show low voltage high resolution scanning electronmicrographs of electrospun mats: (a) individual fiber surface aftermethanol treatment, (b) PEO non-extracted mat, and (c) PEO extractedmat.

FIG. 11 shows representative mechanical properties of electrospunfibers.

FIGS. 12A-12B show phase-contrast microscopy images of BMSCs growing ontissue culture plastic (poly(styrene)) after 1 day of culture in thepresence of (a) PEO non-extracted mats and (b) PEO extracted mats (×40,scale bar: 100 μm).

FIG. 13 shows scanning electron micrographs of BMSCs growing onelectrospun mats and native silk fibroin matrices after 1, 7, and 14days (scale bar: 500 μm).

FIGS. 14A-14D show scanning electron micrographs of BMSCs growing onelectrospun mats after 1 and 14 days: (scale bars: (a) 50 μm, (b) 20 μm,(c) 20 μm, and (d) 10 μm).

FIG. 15 shows proliferation of BMSCs seeded on electrospun mats (seedingdensity: 25,000 cells/cm², N=4). Error bars correspond to the standarddeviations.

FIG. 16 shows MTT results with seeding conditions: 25000/cm², 20% serumafter 14 days. Column heights correspond to the mean values and theerror bars to the standard deviations (n=3).

DETAILED DESCRIPTION OF THE INVENTION

We have developed an all-aqueous process for producing silkbiomaterials, e.g., electrospun silk fibers, films, foams and mats. Thisprocess effectively avoids the problems of (1) poor biocompatibility dueto organic solvents used and (2) embrittled materials associated withconformational transitions of silk protein (e.g., silkworm fibroin)during solubilization and reprocessing from an aqueous solution. Theprocess of the present invention comprises adding a biocompatiblepolymer to an aqueous solution of a silk protein. The solution is thenprocessed to form a silk biomaterial.

The silk protein suitable for use in the present invention is preferablyfibroin or related proteins (i.e., silks from spiders). Preferably,fibroin or related proteins are obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk is obtained,for example, from Bombyx mori. Spider silk may be obtained from Nephilaclavipes. In the alternative, the silk protein suitable for use in thepresent invention can be obtained from a solution containing agenetically engineered silk, such as from bacteria, yeast, mammaliancells, transgenic animals or transgenic plants. See, for example, WO97/08315 and U.S. Pat. No. 5,245,012.

The silk protein solution can be prepared by any conventional methodknown to one skilled in the art. For example, B. mori cocoons are boiledfor about 30 minutes in an aqueous solution. Preferably, the aqueoussolution is about 0.02M Na₂CO₃. The cocoons are rinsed, for example,with water to extract the sericin proteins and the extracted silk isdissolved in an aqueous salt solution. Salts useful for this purposeinclude, lithium bromide, lithium thiocyanate, calcium nitrate or otherchemical capable of solubilizing silk. A strong acid such as formic orhydrochloric may also be used. Preferably, the extracted silk isdissolved in about 9-12 M LiBr solution. The salt is consequentlyremoved using, for example, dialysis.

The biocompatible polymer preferred for use in the present invention isselected from the group comprising polyethylene oxide (PEO) (U.S. Pat.No. 6,302,848) [24], polyethylene glycol (PEG) (U.S. Pat. No.6,395,734), collagen (U.S. Pat. No. 6,127,143), fibronectin (U.S. Pat.No. 5,263,992), keratin (U.S. Pat. No. 6,379,690), polyaspartic acid(U.S. Pat. No. 5,015,476), polylysine (U.S. Pat. No. 4,806,355),alginate (U.S. Pat. No. 6,372,244), chitosan (U.S. Pat. No. 6,310,188),chitin (U.S. Pat. No. 5,093,489), hyaluronic acid (U.S. Pat. No.387,413), pectin (U.S. Pat. No. 6,325,810), polycaprolactone (U.S. Pat.No. 6,337,198), polylactic acid (U.S. Pat. No. 6,267,776), polyglycolicacid (U.S. Pat. No. 5,576,881), polyhydroxyalkanoates (U.S. Pat. No.6,245,537), dextrans (U.S. Pat. No. 5,902,800), polyanhydrides (U.S.Pat. No. 5,270,419), and other biocompatible polymers. Preferably, thePEO has a molecular weight from 400,000 to 2,000,000 g/mol. Morepreferably, the molecular weight of the PEO is about 900,000 g/mol. Ascontemplated by the present invention, two or more biocompatiblepolymers can be directly added to the aqueous solution simultaneously.

The present invention, in one embodiment, provides a fiber produced by aprocess of preparing an aqueous solution of a silk protein, adding abiocompatible polymer to the aqueous solution, and electrospinning thesolution, thereby forming the fiber. Preferably, the fiber has adiameter in the range from 50 to 1000 nm.

In this embodiment, the aqueous solution preferably has a concentrationof about 0.1 to about 25 weight percent of the silk protein. Morepreferably, the aqueous solution has a concentration of about 1 to about10% weight percent of the silk protein.

While not wishing to be bound by theory, it is believed that theaddition of a biocompatible polymer or a plurality of biocompatiblepolymers described above generates viscosity and surface tensionsuitable for electrospinning.

Electrospinning can be performed by any means known in the art (see, forexample, U.S. Pat. No. 6,110,590). Preferably, a steel capillary tubewith a 10 mm internal diameter tip is mounted on an adjustable,electrically insulated stand. Preferably, the capillary tube ismaintained at a high electric potential and mounted in the parallelplate geometry. The capillary tube is preferably connected to a syringefilled with silk/biocompatible polymer solution. Preferably, a constantvolume flow rate is maintained using a syringe pump, set to keep thesolution at the lip of the tube without dripping. The electricpotential, solution flow rate, and the distance between the capillarytip and the collection screen are adjusted so that a stable jet isobtained. Dry or wet fibers are collected by varying the distancebetween the capillary tip and the collection screen.

A collection screen suitable for collecting silk fibers can be a wiremesh, a polymeric mesh, or a water bath. Alternatively and preferably,the collection screen is an aluminum foil. The aluminum foil can becoated with Teflon fluid to make peeling off the silk fibers easier. Oneskilled in the art will be able to readily select other means ofcollecting the fiber solution as it travels through the electric field.As is described in more detail in the Examples section below, theelectric potential difference between the capillary tip and the aluminumfoil counter electrode is, preferably, gradually increased to about 12kV, however, one skilled in the art should be able to adjust theelectric potential to achieve suitable jet stream.

The process of the present invention may further comprise steps ofimmersing the spun fiber into an alcohol/water solution to inducecrystallization of silk. The composition of alcohol/water solution ispreferably 90/10 (v/v). The alcohol is preferably methanol, ethanol,isopropyl alcohol (2-propanol) or n-butanol. Methanol is most preferred.Additionally, the process may further comprise the step of washing thefibroin fiber in water.

In another embodiment, the biomaterial is a film. The process forforming the film comprises, for example, the steps of (a) preparing anaqueous silk fibroin solution comprising silk protein; (b) adding abiocompatible polymer to the aqueous solution; (c) drying the mixture;and (d) contacting the dried mixture with an alcohol (preferred alcoholsare listed above) and water solution to crystallize a silk blend film.Preferably, the biocompatible polymer is poly(ethylene oxide) (PEO). Theprocess for producing the film may further include step (e) of drawingor mono-axially stretching the resulting silk blend film to alter orenhance its mechanical properties. The stretching of a silk blend filminduces molecular alignment in the fiber structure of the film andthereby improves the mechanical properties of the film [46-49].

In a preferred embodiment, the film comprises from about 50 to about99.99 part by volume aqueous silk protein solution and from about 0.01to about 50 part by volume PEO. Preferably, the resulting silk blendfilm is from about 60 to about 240 μm thick, however, thicker samplescan easily be formed by using larger volumes or by depositing multiplelayers.

In a further embodiment, the biomaterial is a foam. Foams may be madefrom methods known in the art, including, for example, freeze-drying andgas foaming in which water is the solvent or nitrogen or other gas isthe blowing agent, respectively.

In one embodiment, the foam is a micropatterned foam. Micropatternedfoams can be prepared using, for example, the method set forth in U.S.Pat. No. 6,423,252, the disclosure of which is incorporated herein byreference.

For example, the method comprising contacting the silkprotein/biocompatible polymer solution with a surface of a mold, themold comprising on at least one surface thereof a three-dimensionalnegative configuration of a predetermined micropattern to be disposed onand integral with at least one surface of the foam, lyophilizing thesolution while in contact with the micropatterned surface of the mold,thereby providing a lyophilized, micropatterned foam, and removing thelyophilized, micropatterned foam from the mold. Foams prepared accordingthis method comprise a predetermined and designed micropattern on atleast one surface, which pattern is effective to facilitate tissuerepair, ingrowth or regeneration, or is effective to provide delivery ofa protein or a therapeutic agent.

In another embodiment, the biomaterial is a scaffold produced using amolding process. See, for example, WO 03/004254 and WO 03/022319. Usingsuch a process, for example, the silk protein/biocompatible polymersolution is placed into a mold, the mold being a negative of the desiredshape of the scaffold. The solution is cured and removed from the mold.In certain embodiments, it may be desirable to form pores in the polymerusing, for example, particulate leaching and other methods known in theart.

Additional biomaterials may be formed with the composition of thepresent invention using ink jet printing of patterns, dip pennanolithography patterns and microcontact printing. See, Wilran et al.,(2001) PNAS 98:13660-13664 and the references cited therein.

The biomaterials produced by the processes of the present invention maybe used in a variety of medical applications such as wound closuresystems, including vascular wound repair devices, hemostatic dressings,patches and glues, sutures, drug delivery and in tissue engineeringapplications, such as, for example, scaffolding, ligament prostheticdevices and in products for long-term or bio-degradable implantationinto the human body. A preferred tissue engineered scaffold is anon-woven network of electrospun fibers.

Additionally, these biomaterials can be used for organ repairreplacement or regeneration strategies that may benefit from theseunique scaffolds, including but are not limited to, spine disc, cranialtissue, dura, nerve tissue, liver, pancreas, kidney, bladder, spleen,cardiac muscle, skeletal muscle, tendons, ligaments and breast tissues.

In another embodiment of the present invention, silk biomaterials cancontain therapeutic agents. To form these materials, the polymer wouldbe mixed with a therapeutic agent prior to forming the material orloaded into the material after it is formed. The variety of differenttherapeutic agents that can be used in conjunction with the biomaterialsof the present invention is vast. In general, therapeutic agents whichmay be administered via the pharmaceutical compositions of the inventioninclude, without limitation: antiinfectives such as antibiotics andantiviral agents; chemotherapeutic agents (i.e. anticancer agents);anti-rejection agents; analgesics and analgesic combinations;anti-inflammatory agents; hormones such as steroids; growth factors(bone morphogenic proteins (i.e. BMP's 1-7), bone morphogenic-likeproteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal growth factor (EGF),fibroblast growth factor (i.e. FGF 1-9), platelet derived growth factor(PDGF), insulin like growth factor (IGF-I and IGF-II), transforminggrowth factors (i.e. TGF-.beta.I-III), vascular endothelial growthfactor (VEGF)); and other naturally derived or genetically engineeredproteins, polysaccharides, glycoproteins, or lipoproteins. These growthfactors are described in The Cellular and Molecular Basis of BoneFormation and Repair by Vicki Rosen and R. Scott Thies, published by R.G. Landes Company hereby incorporated herein by reference.

Silk biomaterials containing bioactive materials may be formulated bymixing one or more therapeutic agents with the polymer used to make thematerial. Alternatively, a therapeutic agent could be coated on to thematerial preferably with a pharmaceutically acceptable carrier. Anypharmaceutical carrier can be used that does not dissolve the foam. Thetherapeutic agents, may be present as a liquid, a finely divided solid,or any other appropriate physical form. Typically, but optionally, thematrix will include one or more additives, such as diluents, carriers,excipients, stabilizers or the like.

The amount of therapeutic agent will depend on the particular drug beingemployed and medical condition being treated. Typically, the amount ofdrug represents about 0.001 percent to about 70 percent, more typicallyabout 0.001 percent to about 50 percent, most typically about 0.001percent to about 20 percent by weight of the material. Upon contact withbody fluids the drug will be released.

The biocompatible polymer may be extracted from the biomaterial prior touse. This is particularly desirable for tissue engineering applications.Extraction of the biocompatible polymer may be accomplished, forexample, by soaking the biomaterial in water prior to use.

The tissue engineering scaffolds biomaterials can be further modifiedafter fabrication. For example, the scaffolds can be coated withbioactive substances that function as receptors or chemoattractors for adesired population of cells. The coating can be applied throughabsorption or chemical bonding.

Additives suitable for use with the present invention includebiologically or pharmaceutically active compounds. Examples ofbiologically active compounds include cell attachment mediators, such asthe peptide containing variations of the “RGD” integrin binding sequenceknown to affect cellular attachment, biologically active ligands, andsubstances that enhance or exclude particular varieties of cellular ortissue ingrowth. Such substances include, for example, osteoinductivesubstances, such as bone morphogenic proteins (BMP), epidermal growthfactor (EGF), fibroblast growth factor (FGF), platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor (IGF-I and II), TGF-, YIGSR peptides, glycosaminoglycans(GAGs), hyaluronic acid (HA), integrins, selectins and cadherins.

The scaffolds are shaped into articles for tissue engineering and tissueguided regeneration applications, including reconstructive surgery. Thestructure of the scaffold allows generous cellular ingrowth, eliminatingthe need for cellular preseeding. The scaffolds may also be molded toform external scaffolding for the support of in vitro culturing of cellsfor the creation of external support organs.

The scaffold functions to mimic the extracellular matrices (ECM) of thebody. The scaffold serves as both a physical support and an adhesivesubstrate for isolated cells during in vitro culture and subsequentimplantation. As the transplanted cell populations grow and the cellsfunction normally, they begin to secrete their own ECM support.

In the reconstruction of structural tissues like cartilage and bone,tissue shape is integral to function, requiring the molding of thescaffold into articles of varying thickness and shape. Any crevices,apertures or refinements desired in the three-dimensional structure canbe created by removing portions of the matrix with scissors, a scalpel,a laser beam or any other cutting instrument. Scaffold applicationsinclude the regeneration of tissues such as nervous, musculoskeletal,cartilaginous, tendenous, hepatic, pancreatic, ocular, integumenary,arteriovenous, urinary or any other tissue forming solid or holloworgans.

The scaffold may also be used in transplantation as a matrix fordissociated cells, e.g., chondrocytes or hepatocytes, to create athree-dimensional tissue or organ. Any type of cell can be added to thescaffold for culturing and possible implantation, including cells of themuscular and skeletal systems, such as chondrocytes, fibroblasts, musclecells and osteocytes, parenchymal cells such as hepatocytes, pancreaticcells (including Islet cells), cells of intestinal origin, and othercells such as nerve cells, bone marrow cells, skin cells, pluripotentcells and stem cells, and combination thereof, either as obtained fromdonors, from established cell culture lines, or even before or aftergenetic engineering. Pieces of tissue can also be used, which mayprovide a number of different cell types in the same structure.

The cells are obtained from a suitable donor, or the patient into whichthey are to be implanted, dissociated using standard techniques andseeded onto and into the scaffold. In vitro culturing optionally may beperformed prior to implantation. Alternatively, the scaffold isimplanted, allowed to vascularize, then cells are injected into thescaffold. Methods and reagents for culturing cells in vitro andimplantation of a tissue scaffold are known to those skilled in the art.

The biomaterials of the present intention may be sterilized usingconventional sterilization process such as radiation based sterilization(i.e. gamma-ray), chemical based sterilization (ethylene oxide) or otherappropriate procedures. Preferably the sterilization process will bewith ethylene oxide at a temperature between 52-55° C. for a time of 8hours or less. After sterilization the biomaterials may be packaged inan appropriate sterilize moisture resistant package for shipment and usein hospitals and other health care facilities.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the invention.

EXAMPLES Example I Materials

Cocoons of B. mori silkworm silk supplied by Institute of Sericulture,Tsukuba, Japan. PEO with an average molecular weight of 4×105 g/mol and9×105 g/mol (Aldrich) were used in blending.

Preparation of Regenerated B. mori Silk Fibroin Solutions

B. mori silk fibroin was prepared as follows as a modification of ourearlier procedure [25]. Cocoons were boiled for 30 min in an aqueoussolution of 0.02 M Na2CO3, then rinsed thoroughly with water to extractthe glue-like sericin proteins. The extracted silk was then dissolved in12 M LiBr solution at 60° C. yielding a 20% (w/v) solution. Thissolution was dialyzed in water using a Slide-a-Lyzer dialysis cassette(Pierce, MWCO 2000). The final concentration of aqueous silk solutionwas 3.0 to 7.2 wt %, which was determined by weighing the remainingsolid after drying. HFIP silk solution (1.5 wt %) was prepared bydissolving the silk fibroin produced after lyophilizing the aqueous silksolution into the HFIP.

Preparation of Spinning Solutions

Silk/PEO blends in water were prepared by adding PEO (900,000 g/mol)directly into the silk aqueous solutions generating 4.8 to 8.8 wt %silk/PEO solutions. Silk solution in HFIP (1.5 wt %) and PEO (4.0 wt %)solution in water, respectively, were also prepared as control solutionsfor comparisons with the blend systems. Silk solution in HFIP wasprepared by dissolving the lyophilized silk fibroin in HFIP at roomtemperature. The viscosity and conductivity of the solutions weremeasured with a Couette viscometer (Bohlin V88) with a shear rate from24.3 to 1216 per second, and a Cole-Parmer conductivity meter (19820) atroom temperature, respectively.

Electrospinning

Electrospinning was performed with a steel capillary tube with a 1.0 mminside diameter tip mounted on an adjustable, electrically insulatedstand. The capillary tube was maintained at a high electric potentialfor electrospinning and mounted in the parallel plate geometry. Thecapillary tube was connected to a syringe filled with 10 ml of asilk/PEO blend or silk solution. A constant volume flow rate wasmaintained using a syringe pump, set to keep the solution at the tip ofthe tube without dripping. The electric potential, solution flow rate,and the distance between the capillary tip and the collection screenwere adjusted so that a stable jet was obtained. By varying the distancebetween the capillary tip and the collection screen, either dry or wetfibers were collected on the screen.

Solution Treatment of Electrospun Mat from Silk/PEO Blend Solutions

Electrospun non-woven mats from silk/PEO blend solutions were immersedinto a 90/10 (v/v) methanol/water solution for 10 min to induce anamorphous to β-sheet conformational transition of electrospun silk fiberand then washed with water for 24 hours at room temperature and 36.5°C., respectively, to remove PEO electrospun fiber from the mats.

SEM

Images of electrospun fibers were obtained with a LEO Gemini 982 FieldEmission Gun SEM.

FT-IR

The infrared spectra were measured with a ATR-FTIR (Bruker Equinox 55)spectrophotometer. Each spectra for samples was acquired intransmittance mode on ZnSe ATR crystal cell by accumulation of 256 scanswith a resolution of 4 cm⁻¹ and a spectral range of 4000-600 cm⁻¹.

XPS

A Surface Science Inc. Model SSX-100 X-ray photoelectron spectrometerwas used to analyze the surface of the silk films to estimate thesurface density of peptides. Survey scans (spot 1000 μm, resolution 4,window 1000 eV) were performed using a flood gun (charge neutralizer)setting of 5 eV and nickel wire mesh held over the sample to preventcharging of the sample surface.

Properties of Silk/PEO Blend Solutions with Pure Silk and PEO Solutions

Aqueous silk solutions without PEO did not electrospin; no fibers wereformed because the viscosity and surface tension of the solution werenot high enough to maintain a stable drop at the end of the capillarytip. Higher concentrations of silk in water to increase the viscosity ofthe solution resulted in gel formation. A stable drop at the end of thecapillary tip was achieved once the PEO was added to the silk solutionat the ratio shown in Table 1. The viscosity of pure silk solution wasmuch lower than other solutions, even at a high concentration of 7.2% asshown in FIG. 1. A small portion of PEO in the silk solution increasedthe viscosity of the blends. The viscosities of silk/PEO blend solutionsdepended on the amount of PEO. The conductivities of silk and silk/PEOblend solutions were higher than pure PEO solutions at room temperature.All silk/PEO blend solutions showed good properties related to viscosityand conductivity in order to electrospin.

Fiber Formation and Morphology of Electrospun Silk/PEO from WaterSolutions

The addition of PEO to silk solutions generated a viscosity and surfacetension suitable for electrospinning. Aluminum foils was used as thecollection screen. As the potential difference between the capillary tipand the aluminum foil counter electrode was gradually increased to 12kV, the drop at the end of the capillary tip elongated from ahemispherical shape into a cone shape, often referred to as a Taylorcone. The applied 12 kV resulted in a jet being initiated near the endof the capillary tip. The distance between the tip and the collector was200 mm and flow rate of all fluid was 0.02 ml/min to 0.05 ml/min. Beforeall solutions were electrospun, Teflon fluid was deposited on collectionscreen to peel off the mat easily.

The morphology and diameters of the electrospun fibers produced wereexamined using high resolution low voltage SEM. All silk/PEO blendsolutions produced fine uniform fibers with less than 800 nm averagefiber diameters (Table 1).

The fiber size was compared between sericin extracted silkworm silk andelectrospun fibers (No. 6) (FIG. 2). The size of electrospun fiber was40 times smaller than the native silk fiber. The individual electrospunfibers appeared to be randomly distributed in the non-woven mat. Amicrograph of the electrospun fibers from a silk/PEO solution in waterare shown in FIGS. 3A-3D.

XPS was used to estimate the surface composition of the mats. Table 2shows the respective peak intensities of O1s, C1s or N1s of PEO, silkfibroin and silk/PEO blends from electrospun mats. The ratios of N1s/C1sand O1s/C1s of the silk mat were 0.31 and 0.40, respectively. In thecase of the silk/PEO mats, N1s/C1s decreased to 0.16 at minimum andO1s/C1s increased to 0.49 at maximum. Based on these ratios we canestimate the fiber composition as shown in Table 2.

Solvent Treatment of Electrospun Mats

The mat was contacted with a 90/10 (v/v) methanol/water solution for 10minutes to induce crystallization of silk and then stored in warm waterat 36.5° C. for 24 hours to extract PEO. The structure change of silkfiber between just elecrospun fiber and fiber after methanol treatmentwas observed by ATR-FTIR. As shown in FIG. 4, its structure was randomcoil or silk I, when it was just electrospun. So it was easily solublein water and lost fiber structure quickly. But, after methanoltreatment, its structure was changed into beta-sheet in FIG. 4. So, evenafter it was stored in water, it showed fine fiber structure.

XPS was used to analyze the surface of the mat after methanol/watertreatment and washing with water to estimate the surface composition.Table 2 shows the XPS spectra results of PEO, silk fibroin and silk/PEOblend electrospun mats. Their respective peak intensities of O1s, C1s orN1s are also shown in Table 2. The ratio of N1s and C1s of all blend matwas less than the silk mat (0.33) even after washing with water.Therefore the individual silk/PEO electrospun fibers have PEO phasesinside. Based on these ratios we can estimate the composition of thesurface of the mat, relative of the solution used in spinning.

Example II Materials

Cocoons of B. mori silkworm silk were obtained from M Tsukada, Instituteof Sericulture, Tsukuba, Japan. PEO with an average molecular weight of9×105 g/mol and polyethylene glycol (PEG) (3,400 g/mol) were purchasedfrom Aldrich and used without further purification.

Preparation of Regenerated B. mori Silk Fibroin Solutions

B. mori silk fibroin solutions were prepared by modifying the proceduredescribed earlier [25]. Cocoons were boiled for 30 min in an aqueoussolution of 0.02 M Na2CO3, then rinsed thoroughly with water to extractthe glue-like sericin proteins. The extracted silk was then dissolved in9.3 M LiBr solution at room temperature yielding a 20 wt % solution.This solution was dialyzed in water using a Slide-a-Lyzer dialysiscassette (Pierce, MWCO 2000) for 48 hrs. The final concentration ofaqueous silk solution was 7.0 to 8.0 wt %, which was determined byweighing the remaining solid after drying.

Preparation and Treatment of Blend Films

Various silk blends in water were prepared by adding 4 wt % PEG or PEOsolutions into the silk aqueous solutions. The blending ratios (silk/PEGor PEO) were 100/0, 95/5, 90/10, 80/20, 70/30 and 60/40 (w/w). Thesolutions were mildly stirred for 15 min at room temperature and thencast on polystyrene Petri dish surfaces for 24 hrs at room temperaturein a hood. The films then placed vacuum for another 24 hrs. Silk fibroinand blend films were immersed in a 90/10 (v/v) methanol/water solutionfor 30 min to induce an amorphous to β-sheet conformational transitionof the silk fibroin. After crystallizing the silk and silk/PEG or PEOblends using methanol, their solubility in water, 17 MΩ at 37° C., wasdetermined for 48 hrs. This solubility test was performed in shakingincubator and shaking speed was 200 rpm. It was expected that just PEGor PEO would dissolve. Solubility was calculated by weight balancebetween before and after PEG or PEO extraction.

Characterization

Fractured surfaces of silk and silk/PEG or PEO blend films were imagedusing a LEO Gemini 982 Field Emission Gun SEM. A Surface Science Inc.Model SSX-100 X-ray photoelectron spectrometer was used to analyze thesurface of the silk films to estimate the surface density of silkpeptide versus PEO. Survey scans (spot 1000 μm, resolution 4, window1000 eV) were performed using a flood gun (charge neutralizer) settingof 5 eV and nickel wire mesh held over the sample to prevent charging ofthe sample surface.

A differential scanning calorimeter, DSC (2920 Modulated DSC) from TAInstruments, was utilized to determine the thermal properties of thesilk and blended films. Indium was used to calibrate temperature and thesample was sealed in aluminum pan. Each scans were performed −20° C. to320° C. with a rate of 10° C./min. Sample were cooled to −100° C. at 20°C./min.

Contact Angle Analysis

The contact angle using Millipore purified water droplet, 17 MΩ, on thesilk and blend films was measured to determine surface hydrophilicity.The water droplet, approximately 5 μl, was applied using a syringe and22-gauge needle, and the static contact angle measured using agoniometer (Rame-Hart, Inc.). This analysis was performed after methanoltreatment.

Mechanical Properties of Silk and Blend Films

The tensile properties of specimens (5×50×0.2 mm) were measured with acrosshead speed of 15 mm/min using Instron tensile tester at ambientcondition. Gauge length was set 30 mm and initial load cell of 100 kgfwas applied. The tensile strength per cross-sectional area (kg/mm2) andthe ratio of the relative elongation to the initial film length at break(%) were determined from an observation of the stress-strain curves.

Blending Silk with PEG or PEO

PEG and PEO were selected for blending with silk to improve silk filmproperties with aqueous processability and biocompatibility as keycriteria. PEG or PEO were studied for blending (with molecular weightsof 3,400 and 900,000 g/mol, respectively). Silk/PEG or PEO films werefirst prepared to identify concentrations of the components useful inmaterials processing. The films were cast from water solutions ontopolystyrene Petri dish in various ratios (Table 4) and dried overnight.In the case of silk and PEG (3,400 g/mol) blends, the two componentsseparated macroscopically into two phases during film formationthroughout the range of compositions studied. Poorer quality filmsformed from all blend ratios except silk/PEG (98/2). Blends fromsilk/PEG were immersed in a 90/10 (v/v) methanol/water solution for 30min to content the fibroin to the insoluble β-sheet structure. Afterthis crystallization process, phase separation was more pronouncedbecause the PEG phases became opaque while the silk phase was stilltransparent. Because the phase separation in the silk/PEG (60/40) blendwas the most pronounced, further characterization was not considered onsilk/PEG (60/40) blends. However, in the case of silk and PEO (900,000g/mol) blends, no macroscopic phase separation occurred between twocomponents throughout the range of components studied.

Aqueous Solubility of Blend Films

Solubility was calculated by weight balance between before and after PEOor PEG extraction, as shown in Table 5. Silk or blend films wereseparated into 6 parts, 3 parts of which were put into 3 independentglass vials for solubility testing at 12, 24 and 48 hrs. Up to 48 hrs,pure silk fibroin films did not show significant weight loss since theyhad been crystallized in methanol for 30 min before solubility testing.The slight weight change (˜0.6%) during the test was believed to be dueto the subtle effects of physical shear due to the vigorous shaking.Errors in the range of 1% were considered insignificant throughout thestudy. FIGS. 5A-5B showed the percentage weight loss of silk andsilk/PEO or PEG blends according to time. In the case of silk/PEOblends, they showed relatively even weight loss throughout the range ofcompositions due to water solubility of PEO in the blends (FIG. 5A).

DSC

Thermal properties of silk and silk/PEO blends were observed by DSCbefore and after methanol treatment (FIGS. 6 and 7). DSC thermograms ofthe regenerated silk films are shown in FIG. 6( a) and FIG. 7( a), whichare before and after methanol treatment, respectively. FIG. 6( a) showsan exothermic peak at about 88.2° C., attributed to the crystallizationof silk fibroin induced by heat, and three endotherms at around 54.2,137.7 and 277.9° C., attributed to the glass transition temperature,water evaporation and thermal degradation of silk fibroin, respectively[50]. On the other hand, FIG. 7( a) shows an endotherm at 278° C.without any trace of exothermic transition. This behavior is due tobeta-sheet structure formation of silk film during methanol treatment[51].

In FIG. 6, overlapping of the characteristic thermal transitions of silkfibroin and PEO from 54.2 to 64.2° C. in blends seems the main featureemerging from the above DSC results. However, some changes appearing inthe DSC pattern of blend films with high PEO content (more than 20 wt %)may suggest that a certain degree of interaction was established betweensilk fibroin and PEO. We mainly refer to the shift to lower temperatureof the peak of PEO melting temperature and the disappearance of thecrystallization peak of silk at 87 to 88° C., as well, with increasingPEO content in the blends. These effects can be interpreted as adecrease of PEO crystallization temperature in the blends and aprevention of the silk crystallization after PEO melts in the blends,due to the interaction of silk and PEO molecules. Otherwise, aftermethanol treatment of all samples, FIG. 7 shows the melting temperatureof PEO in the blends shifted just slightly because of mostly the phaseseparation between silk and PEO domain by the crystallisation of silk.As it is shown in SEM observation as following, the two componentsformed micro phase separation in the blends.

Even though the maximum thermal degradation temperature seems to be lessaffected by methanol treatments and blending with PEO and its ratio,some changes were observed, such as a slight broadening of thedecomposition endotherm with increasing the amount of PEO on the blendin the case of blends before methanol treatment.

XPS

XPS was used to estimate the surface composition of the films. Table 6shows the respective peak intensities of O1s, C1s or N1s of silk fibroinand silk/PEO blend films before and after methanol treatments. Theratios of N1s/C1s were used to estimate the composition of silk and PEObefore and after methanol treatments from the surface of films. Based onthese ratios we can estimate the blend film composition as shown inTable 6. As the PEO portion was increased, the N1s/C1s of all blends wasdecreased in both of before and after methanol treatment. Especially,the N1s/C1s after methanol treatment on blend films was much lower thanbefore methanol treatment. It could be estimated that the PEO partmigrates into the surface of film by phase separation during methanoltreatment, because of silk β-sheet formation. Since silk is relativelyhydrophobic, it might be anticipated a lower content of silk on the filmsurface treated in methanol could be anticipated. However, the N1s/C1sratio of silk/PEO (90/10) was increased after methanol treatment

SEM

The fractured cross section side and surface morphologies of the silkand silk/PEO or PEG blend films were examined using high resolution lowvoltage SEM after PEG or PEO extraction in warm water at 37 for 48 hrs.While the pure silk fibroin film exhibited a dense and uniformmicrostructure, the fractured surfaces of all silk/PEO blends showed arough morphology due to the micro phase separation. The higher the PEOcontent in the films up to 40 wt %, the denser the film morphology basedon cross sections. The silk/PEO (90/10) blend showed the least densemorphology from the fractured surfaces, which demonstrates that the PEOportion of the blend does not migrate to the surface during methanoltreatment. This conclusion supports the SPS data. The silk/PEG blendfilms, unlike the silk PEO systems, did not show a different morphologythan that seen with the pure silk fibroin films.

Contact Angle Measurements

The contact angle was measured on the silk and silk/PEO blend filmsafter methanol treatment as shown in Table 7. The hydrophilicity ofsurface was increased with increasing the PEO ratio of the blend.

Mechanical Properties

The values of tensile modulus, rupture strength and elongation of silkand silk/PEO blend films are shown in Table 8. The pure silk filmdisplayed the typical behavior of brittle materials. The addition of 2wt % PEO to silk fibroin was effective in inducing a slight improvementof the mechanical properties of blend films. In other ratios of blend,tensile modulus and strength decreased with increasing the PEO content.However, elongation at break was increased slightly up to 10.9% insilk/PEO (60/40) blends. Methanol treatment of these samples did notsignificantly change the mechanical properties.

Drawing (Stretching) of Silk Blend Film

PEO02BM (silk/PEO 98/02 wt %) film blend sample was soaked in water for5 minutes at room temperature and then stretched two times its originallength. Then, the sample was dried at ambient conditions for 48 hrsfollowed by tensile testing on an Instron.

Tensile Modulus Tensile Strength Elongation at Samples (GPa) (MPa) Break(%) PEO02BM¹ 3.3 63 5.7 PEO02ST² 2.3 88 41 ¹BM: before methanoltreatment, ²ST: stretched

Example III Methods

Cocoons of B. mori silkworm silk were kindly supplied by M. Tsukada,Institute of Sericulture, Tsukuba, Japan. PEO with an average molecularweight of 9×105 g/mol (Aldrich) was used in the blends.

Preparation of Silk Matrices and Regenerated B. Mori Silk FibroinSolutions

To prepare the silk matrices for cell seeding experiments, whiteBrazilian raw Bombyx mori silkworm fibers were extracted for 1 hour at90° C. in an aqueous solution of 0.02 M Na2CO3 and 0.3% (w/v) detergentas previously described [33] to remove sericin, the antigenic glue-likeproteins that encapsulate the fibroin fibers following secretion fromthe silkworm. A 3-cm long silk wire-rope matrices consisting of 540 silkfibers (pre-extraction) were generated for use in this study by crimpingends with stainless steel 316L collars (1 cm in length, 2.2 mm I.D, 3 mmO.D.).

Regenerated B. mori silk fibroin solutions was prepared as amodification of our earlier procedure. Cocoons were boiled for 30 mm inan aqueous solution of 0.02 M Na2CO3, and then rinsed thoroughly withwater to extract sericin proteins [25]. The extracted silk was thendissolved in 9.3 M LiBr solution at 60° C. yielding a 20% (w/v)solution. This solution was dialyzed in water using a Slide-a-Lyzerdialysis cassette (Pierce, MWCO 3500). The final concentration ofaqueous silk solution was 8.0 wt %, which was determined by weighing theremaining solid after drying.

Preparation of Spinning Solutions

Silk/PEO blends (80/20 wt/wt) in water were prepared by adding 5 ml of5.0 wt % PEO (900,000 g/mol) into 20 ml of 8 wt % silk aqueous solutiongenerating 7.5 wt % silk/PEO solutions. To avoid the premature formationof β-sheet structure during blending the two solutions, the solutionswere stirred gently at low temperature, 4° C.

Electrospinning

Electrospinning was performed with a steel capillary tube with a 1.5 mminside diameter tip mounted on an adjustable, electrically insulatedstand as described earlier [9, 32]. The capillary tube was maintained ata high electric potential for electrospinning and mounted in theparallel plate geometry. The capillary tube was connected to a syringefilled with 10 ml of a silk/PEO blend solution. A constant volume flowrate was maintained using a syringe pump, set to keep the solution atthe tip of the tube without dripping. The electric potential, solutionflow rate, and the distance between the capillary tip and the collectionscreen were adjusted so that a stable jet was obtained. By varying thedistance between the capillary tip and the collection screen, either dryor wet fibers were collected on the screen.

Treatment of Electrospun Mats

Electrospun non-woven mats from silk/PEO blend solutions were immersedinto a 90/10 (v/v) methanol/water solution for 10 mm to induce anamorphous to silk β-sheet conformational transition, and then washedwith water for 48 hours at 37° C. to remove PEO from the mats. Thisprocess was performed in a shaking incubator at 50 rpm. Two sets ofelectrospun mats were studied for cell interactions, with and withoutPEO present.

XPS

A Surface Science Inc. Model SSX-100 X-ray photoelectron spectrometerwas used to analyze the surface of the silk films to estimate thesurface density of silk versus PEO. Survey scans (spot 1000 μm,resolution 4, window 1000 eV) were performed using a flood gun (chargeneutralizer) setting of 5 eV and nickel wire mesh held over the sampleto prevent charging of the sample surface.

DSC

A differential scanning calorimeter (DSC) (2920 Modulated DSC) from TAInstruments was utilized to determine the thermal properties of theelectrospun fibers. Indium was used to calibrate temperature and thesample was sealed in an aluminum pan. Each scan was performed between−20° C. to 100° C. with a rate of 10° C./mm.

Optical Polarizing Microscopy

A Zeiss Axioplan 2 with digital camera and Linkam. LTS 120 hot stage wasused to observe the morphologies of the electrospun fiber. The imageswere taken and compared before heating the fiber at room temperature andafter heating to 100° C. at a rate of 5° C.

Mechanical Properties of Electrospun Mats

The mechanical properties of specimens (8×40×0.5) (mm) were measuredwith a crosshead speed of 20 mm/mm using an Instron tensile tester atambient condition. Gauge length was set at 20 mm and an load cell of 100kg f was used. The tensile strength per cross-sectional area (kg/mm²)and the ratio of the relative elongation to the initial film length atbreak (%) were determined from an observation of the stress-straincurves. All samples were stored in vacuum at room temperature beforetest. Each test was performed 5 times.

Cells and Matrix Seeding

BMSCs were isolated, cultured expanded and stored as previouslydescribed [33]. Briefly, human unprocessed whole bone marrow aspirateswere obtained from donors <25 years of age (Clonetic-Poietics,Walkersville, Md.), resuspended in Dulbecco Modified Eagle Medium (DMEM)supplement with 10% fetal bovine serum (FBS), 0.1 mM nonessential aminoacids, 100 U/ml penicillin and 100 mg/L streptomycin (P/S), and 1 ng/mlbasic fibroblast growth factor (bFGF) and plated at 8 μl aspirate/cm2 intissue culture polystyrene; non-adherent hematopoietic cells wereremoved with the culture medium during medium exchange after 4 days.Thereafter, medium was changed twice a week. Primary BMSCs were detachedprior to confluency using 0.25% trypsin/1 mM EDTA and replated at 5×103cells/cm2. First passage (P1) hBMSCs near confluency were trypsinizedand frozen in 8% DMSO/IO % FBS/DMEM for future use.

Frozen P1 hBMSCs were defrosted and replated at 5×10³ cells/cm2 (P2),trypsinized when near confluency, and used for matrix seeding.Electrospun fibroin mats (1 cm×1 cm) were incubated with 70% alcohol for30 minutes followed by an extensive washing procedure with sterile PBSbefore cell seeding. Matrices were seeded with cells (25000 cells/cm2)by direct pipetting of the cell suspension onto the silk matrices andincubated at 37° C./5% CO₂ in 2 ml of cell culture medium without bFGFfor the duration of the experiment. The cell culture medium was changedevery 4 days.

For seeding BMSCs to silk native fiber matrices, gas sterilized(ethylene oxide) silk matrices (3 cm in length) were placed in a customdesigned Teflon seeding chamber to increase cell-matrix interaction. Thechamber has twenty-four wells, each 3.2 mm wide by 8 mm deep by 40 mmlong (1 ml total volume). Matrices were inoculated with 1 ml of cellsuspension at a concentration of 2×106 cells/ml by direct pipetting,incubated for 2 hours at 37° C./5% CO₂ and transferred to tissue cultureflasks for the duration of the experiment in an appropriate amount ofcell culture medium without bFGF. Following seeding, the silk matriceswere cultured in an appropriate amount of DMEM (10% FBS) for 1 day and14 days.

Cell Proliferation Assays Cell Counting

After 1, 7 and 14 days, the silk mats were harvested, washed with PBS toremove non-adherent cells, then incubated in 0.5 ml of 0.25% typsin/1 mMEDTA at 37° C. for 5 minutes. The trypsinization was stopped by adding0.5 ml of culture medium containing 10% FBS to each sample. The cellnumbers were then counted by using a hematocytometer and microscope.

MTT

Cell proliferation was measured by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)(Sigma, St. Louis, Mo.) staining. After 14 days, seeded silk matrices orsilk mats were incubated in MIT solution (0.5 mg/ml, 37° C./5% CO2) for2 hours. The intense red colored formazan derivatives formed wasdissolved and the absorbance was measured with a microplatespectrophotometer (Spectra Max 250, Molecular Devices, Inc, Sunnyvale,Calif.) at 570 nm and the reference wavelength of 690 nm.

Scanning Electron Microscopy (SEM)

SEM was used to determine cell morphology seeded on the silk fibroin.

Following harvest, seeded silk matrices were immediately rinsed in 0.2 Msodium cacodylate buffer, fixed in Karnovsky fixative (2.5%glutaraldehyde in 0.1 M sodium cacodylate) overnight at 4° C. Fixedsamples were dehydrated through exposure to a gradient of alcoholfollowed by Freon (1,1,2-trichlorotrifluoroethane, Aldrich, Milwaukee,USA) and allowed to air dry in a fume hood. Specimens were examinedusing LEO Gemini 982 Field Emission Gun SEM (high resolution low voltageSEM) and JEOL JSM-840A SEM.

Results and Discussion Electrospinning of Silk/PEO Solutions

In order to increase the viscosity of aqueous silk solution (8 wt %),PEO (MW 900K) was added with the ratios of 4/1 (silk/PEO wt/wt) shown inTable 9 as described above and in our previous work [32]. The viscosityand surface tension of the pure silk solution (8 wt %) were not highenough to maintain a stable drop at the end of the capillary tip. Theaddition of PEO to silk solutions generated a viscosity and surfacetension suitable for electrospinning. The distance between the tip andthe collector was 21.5 cm and flow rate of the fluid was 0.03 ml/min. Asthe potential difference between the capillary tip and the aluminum foilcounter electrode was gradually increased 12.5 kV (E=0.6 kV/cm), thedrop at the end of the capillary tip elongated from a hemisphericalshape into a cone shape. The morphology and diameters of the electrospunfibers were examined using SEM. Silk/PEO blend solution produced fineuniform fibers with 700 nm±50 average fiber diameters (Table 9). Theindividual electrospun fibers appeared to be randomly distributed in thenon-woven mat.

The electrospun fibers from the blend solution were observed by opticalmicroscopy with a hot stage. The melting temperature of PEO is around60° C. [78] and silk fibroins do not show any thermal transitions up to100° C. [79]. FIG. 8( a) was taken at room temperature and FIG. 8( b)after heating to 100° C. at a rate of 5° C./min. This result confirmsthat both polymer (PEO and silk fibroin) were presenting single in theelectrospun fibers. The fact that fibers remained intact in bothtemperatures shows that the melt out of PEO didn't have any effect ontheir morphology and structure. Therefore, the fiber integrity dependsonly on silk fibroin.

Electrospun mats were treated with methanol to eliminate solubility inwater. The surface composition of the mats before and after methanoltreatment was determined by XPS (Table 3). The respective peakintensities of O1S, C1S or N1S of two silk/PEO blends from electrospunmats are illustrated. The ratios of N1S/C1S of the mat was 0.23 beforemethanol treatment. After methanol treatment, the N1S/C1S increased to0.28 (Table 3) as expected due to solubility of PEO in methanol. WhenPEO was extracted from the mat at 37° C. in water for 2 days, theN1S/C1S increased to 0.31, which did not change even after 7 days.Therefore, after the PEO extraction during 2 days, almost all the PEOhad been extracted. DSC measurements confirmed the elimination of PEO bythis treatment as well (FIG. 9). After methanol treatment, theelectrospun mats showed a melting temperature peak for PEO around 56.5°C. (FIG. 9), after extraction in water the peak was absent. To observedelicate surface morphology of electrospun fiber, high resolution lowvoltage SEM was used without a conductive-coating on the sample. Aftermethanol treatment, a surface morphology of electrospun mat was observedand each individual fiber showed the fibril structure with around 110 nmfrom its surface similar to degummed native silk fiber (FIG. 10 (a))[67]. Even after PEO extraction from the electrospun mats, surfacemorphology was maintained (FIGS. 10 (b) and (c)). Two sets ofelectrospun mats with and without PEO present were compared with nativesilk fibro in fibers for cell interactions.

The values of tensile modulus, strength and elongation of electrospunmats are shown in FIG. 11. The scaffold structure must providesufficient mechanical properties during the process of tissueregeneration. After methanol treatment of electospun mats, its tensilemodulus, tensile strength and elongation values were 624.9±0.9 MPa,13.6±1.4 MPa and 4.0±2.0%, respectively. By β-sheet structure formationof electrospun silk fibroin during methanol treatment [32], its tensilemodulus and strength was higher and elongation was lower than beforemethanol treatment. After PEO extraction from electrospun mats, itsmechanical properties were largely decreased due to brittleness as shownin regenerated silk fibroin films [39]. The existence of PEO waseffective in the improvement of the mechanical properties of electrospunmats. Even though its elongation was decreased after methanol treatment,toughness was much higher before PEO then after PEO extraction.Electrospun silk fibroin mats in this study were comparable with otherbiodegradable electrospun mats using PGA [80], PLGA [81], collagen [82],collagen/PEO blends [83] that were used as scaffolds for tissueregeneration.

Cell Culture Experiments

A tissue engineering scaffold material must support cellular attachmentand growth. To evaluate cellular behavior on the electrospun fibroin,BMSCs were seeded and cultivated on the PEO non-extracted or extractedsamples placed in Petri dishes. At 24 hours after seeding, it wasobserved that the PEO extracted silk mats were surrounded by cellsgrowing on tissue culture plastic. In contrast, few cells were observedaround the non-extracted mats (FIG. 12). This phenomenon may suggestthat at day 1, PEO was released from the non-extracted silk mats whichkept BMSC from attaching to the surrounding area. The cell number fromday 1 showed that 50% more cells were attached to PEO-extracted silkmats when compared with non-extracted silk mats. BMSC attachment to silkmats was confirmed by SEM (FIG. 13). Cells were observed on both PEOextracted and non-extracted mats 1 day after cell seeding, but with ahigher density on the PEO extracted samples which cope with the datafrom cell counting experiment. Presumably the soluble PEO released fromthe non-extracted mats during incubation kept the cells from attachingto the fibers, due to the hydrophilic nature of the PEO [84], whichlimits protein adsorption [85-87]. While the cells on non-extracted matsstayed on the surface of the material (FIG. 14 (a)), some cells migratedunderneath the silk fibers on PEO extracted mats (FIG. 14 (b)). However,after 14 days, the cells grew among fibers and covered the majority ofthe surface on both of the extracted and non-extracted fibers (FIGS. 14(c) and (d)).

In both extracted and non-extracted groups, the cell numbers weresignificantly increased (p<0.01) at day 7 when compared with day 1,which suggests cell growth (FIG. 15). Cell number on PEO extracted matswas significantly higher (p<0.05) by approximately 88% compared to thecell number on the non-extracted silk mats. Most parts of the PEOextracted and non-extracted mats were densely populated with BMSCs after7 days of cultivation; a cell sheet and possible ECM covered thesurfaces as determined by SEM (FIG. 13). This may explain the resultthat after day 7, the cell growth showed a plateau in both groups. Thedifference in cell density at day 7 and day 14 between the PEO-extractedand non-extracted groups may be due to differences in initial cellattachment caused by the existence of PEO. The presence of the PEO didnot affect cell growth, which may be due to the fact that the PEO wasextracted at 37° C. after a few days of incubation in cell culturemedium. Our XPS results suggested that PEO was extracted after incubatedthe silk mats at 37° C. in water for 2 days (Table 3). Parallel seedingexperiments were performed on native silk fibers. BMSCs were seeded onnative silk matrices and cultured for 1 day or 14 days. SEM analysisshowed that a few cells attached on native silk fibers (which have adiameter of ˜15 μm on average) at day 1 (FIG. 13). BMSCs reachedconfluency and appeared to fully cover the silk matrices after 14 daysof cultivation. BMSCs seeded and cultivated on the PEO extracted matswere present at higher densities compared to cells on the non-extractedmats. However, these differences were not significant (p>O.O5) (FIG.16).

Conclusions

Fine fiber mats with fibroin diameter 700±50 nm were formed from aqueousB. mori fibroin by electrospinning with PEO with molecular weight of900,000. PEO supplied good mechanical properties to the electrospunmats, even though, initially, residual PEO inhibited cell adhesion.Within 1˜2 days following PEO extraction, those effects were abolishedand proliferation commenced. After 14 days of incubation, theelectrospun silk mats supported extensive BMSC proliferation and matrixcoverage. The ability of electrospun silk matrices to support BMSCattachment, spreading and growth in vitro, combined with abiocompatibility and biodegradable properties of the silk proteinmatrix, suggest potential use of these biomaterial matrices as scaffoldsfor tissue engineering.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventionwithout departing from the spirit and scope of the invention. Thus, itis intended that the present invention cover the modifications andvariations of this invention provided they come within the scope of theappended claims and their equivalents.

REFERENCES

The references cited below and incorporated throughout the applicationare incorporated herein by reference.

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TABLE 1 Concentrations and Conductivities of Silk, PEO, Silk/PEO Blendsand their Electrospun Fibers Initial Conc. PEO Ratio to Average of SilkSilk Total Conductivity Fiber Solutions (%) (PEO/Silk) Conc. (%) (μS)Diam. Silk 7.2 — 7.2 240.0 — No 1 7.2 1/3 8.8 216.5 800 No 2 7.2 1/4 8.3191.9 600 No 3 6.3 1/4 7.3 185.0 600 No 5 6.0 1/3 7.4 209.0 800 No 6 5.31/3 6.6 182.2 600 No 7 4.1 1/2 5.8 175.1 600 No 8 3.0 2/3 4.8 154.3 800PEO — — 4.0 61.3 450

TABLE 2 High-Resolution XPS Results from the Electrospun Silk, PEO, andSilk/PEO blends Surfaces O1s N1s C1s Binding Binding Binding Silk/PEOElement Energy atom % Energy Atom % Energy atom % N1s/C1s W/w PEO 531.237.4 — — 284.6 62.6 —  0/100 Silk 530.3 24.7 398.4 18.9 284.6 56.4 0.33100/0  No. 1 530.9 27.1 398.5 15.3 284.6 57.6 0.26 79/21 No. 1w20 531.023.6 399.1 17.3 284.6 57.9 0.32 97/3  No. 1w37 531.3 23.7 399.3 284.659.0 0.29 88/12 No. 2 530.9 26.5 398.6 15.0 284.6 58.5 0.26 79/21 No.2w20 530.9 22.4 399.1 17.8 284.6 59.8 0.30 91/9  No. 2w37 531.1 24.0399.2 17.7 284.6 58.3 0.30 91/9  No. 3 531.1 25.2 398.9 16.4 284.6 58.40.28 85/15 No. 3w20 530.7 25.2 399.0 18.2 284.6 56.6 0.32 97/3  No. 3w37531.1 23.2 399.0 16.3 284.6 60.5 0.27 82/18 No. 5 530.8 28.4 398.6 13.8284.6 57.8 0.24 73/27 No. 5w20 531.0 25.1 399.0 16.3 284.6 58.6 0.2885/15 No. 5w37 531.2 23.9 399.3 16.8 284.6 59.3 0.28 85/15 No. 6 530.526.4 398.4 15.8 284.6 57.8 0.27 82/18 No. 6w20 530.1 24.0 398.3 17.6284.6 58.4 0.30 91/9  No. 6w37 531.3 23.2 399.3 17.4 284.6 59.4 0.2988/12 No. 7 530.7 26.4 398.4 14.2 284.6 59.4 0.24 73/27 No. 7w20 530.924.5 399.1 17.7 284.6 57.8 0.31 94/6  No. 7w37 530.9 23.5 398.9 17.9284.6 58.6 0.31 94/6  No. 8 531.1 29.2 398.5 10.1 284.6 60.7 0.17 51/49No. 8w20 530.8 24.2 398.9 16.7 284.6 59.1 0.28 85/15 No. 8w37 530.8 24.9398.5 17.5 284.6 57.6 0.30 91/9 

TABLE 3 High-Resolution XPS Results from the Electrospun Silk/PEO BlendsSurfaces O1s N1s C1s Binding Binding Binding Element Energy (eV) Atom %Energy (eV) Atom % Energy (eV) atom % N1s/C1s BM¹ 530.9 24.3 398.6 14.4284.6 61.3 0.23 AM² 530.9 24.7 398.8 16.4 284.6 58.9 0.28 EX2³ 530.924.4 398.9 17.8 284.6 57.8 0.31 EX7⁴ 530.8 24.1 398.4 18.0 284.6 57.90.31 ¹BM: before methanol treatment, ²AM: after methanol treatment, ³EX:after PEO extraction in water for 2 days, ⁴after PEO extraction in waterfor 7 days.

TABLE 4 Silk fibroin/PEG or silk fibroin/PEO blend composition Silkfibroin PEG stock PEO stock Silk/PEG or Silk/PEG Silk/PEO Blend Ratiostock solution solution solution PEO weight ratio Blend Conc. BlendConc. Silk/PEG or PEO (wt %) (wt %) (wt %) in blend (wt %) (wt %)(wt/wt) 8.0 — — 8.0/0   8.0 8.0 98/2  8.0 10.0 4.0 8.0/0.16 8.0 7.898/2  8.0 10.0 4.0 8.0/0.89 8.2 7.3 90/10 8.0 10.0 4.0 8.0/2.00 8.3 6.780/20 8.0 10.0 4.0 8.0/3.43 8.5 6.1 70/30 8.0 10.0 4.0 8.0/5.33 8.7 5.760/40

TABLE 5 Weights (mg) of silk and silk blend films before and after PEGor PEO extraction at 37° C. for 48 hrs. Silk/PEG or Extraction Silk/PEOSilk/PEG PEO Blend Time (hr) Before After Before After 12 96.2 95.6  —²— 100/0  24 89.2 88.6 — — 48 106.8 105.2  — — 12 82.2  80.6 (80.9)¹ 67.264.2 (65.8) 98/2  24 76.7 75.5 (75.2) 40.4 39.5 (39.6) 48 95.7 91.2(93.8) 45.6 41.4 (44.7) 12 79.4 70.0 (71.5) 66.6 46.9 (59.9) 90/10 2479.1 70.6 (71.2) 74.0 66.4 (66.6) 48 74.1 64.3 (66.7) 76.4 67.9 (68.8)12 59.2 51.1 (47.4) 58.9 49.0 (47.1) 80/20 24 54.1 46.7 (43.3) 58.6 47.4(46.9) 48 53.8 45.5 (43.0) 58.8 46.5 (47.0) 12 66.2 52.2 (46.3) 90.059.7 (63.0) 70/30 24 74.2 58.3 (51.9) 114.2 79.5 (79.9) 48 59.8 45.6(41.9) 69.3 49.2 (48.5) 12 57.8 39.8 (34.7) — — 60/40 24 63.8 42.5(38.3) — — 48 51.7 33.6 (31.0) — — ¹Parenthesis: calculated silk weightfrom the blend films. ²Not measured.

TABLE 6 High-Resolution XPS Results from the Silk, PEO, and Silk/PEOblend film surfaces before and after methanol treatment. O1s N1s C1sBinding Binding Binding Element Energy (eV) Atom % Energy (eV) Atom %Energy (eV) atom % N1s/C1s Silk/PEO Silk 530.3 24.6 398.4 14.9 284.660.5 0.25 100/0  PEO02B 530.9 24.2 398.5 14.4 284.6 61.4 0.23 92/8 PEO10B 530.9 29.0 398.6 10.0 284.6 61.0 0.16 64/36 PEO20B 531.1 25.3398.9 12.4 284.6 62.3 0.20 80/20 PEO30B 530.8 25.1 398.6 13.0 284.6 61.90.21 84/16 PEO40B 530.5 24.6 398.4 12.0 284.6 63.4 0.19 76/24 PEO02A530.7 23.8 398.4 13.4 284.6 62.8 0.21 84/16 PEO10A 531.1 23.7 398.5 11.4284.6 64.9 0.17 68/32 PEO20A 530.5 29.3 398.4 7.4 284.6 63.3 0.12 48/52PEO30A 530.7 18.3 398.4 9.5 284.6 72.2 0.13 52/48 PEO40A 531.1 27.0398.5 6.4 284.6 66.6 0.10 40/60

TABLE 7 Contact angle measurement of silk and silk/PEO blend films aftermethanol treatment Sample SILKAM PEO02AM PEO10AM PEO20AM PEO30AM PEO40AMAngle(°) 81 ± 2 76.2 ± 2 72.2 ± 1 68.0 ± 1 68.0 ± 1 63.3 ± 2

TABLE 8 Mechanical properties of the silk and silk/PEO blend filmsbefore and after methanol treatment Tensile Modulus Tensile StrengthElongation at Sample (GPa) (MPa) Break (%) SILKBM 3.9 ± 0.7 47.2 ± 6.41.9 ± 0.7 PEO02BM 3.3 ± 0.6 63.0 ± 8.7 5.7 ± 2.0 PEO10BM 3.2 ± 0.2 42.5± 2.0 2.7 ± 0.6 PEO20BM 2.7 ± 0.3 28.9 ± 2.8 1.9 ± 0.7 PEO30BM 2.3 ± 0.129.5 ± 0.9 6.2 ± 1.8 PEO40BM  2.0 ± 0.03 32.6 ± 3.4 10.9 ± 4.5  SILKAM3.5 ± 0.9  58.8 ± 16.7 2.1 ± 0.4 PEO02AM 3.4 ± 0.1 58.5 ± 6.5 3.2 ± 1.0PEO10BM 3.2 ± 0.1 43.3 ± 4.7 2.6 ± 0.3 PEO20BM 2.3 ± 0.2 27.9 ± 3.0 2.1± 0.2 PEO30BM 2.1 ± 0.2 29.2 ± 5.3 4.9 ± 1.6 PEO40BM 1.4 ± 0.2 26.5 ±2.3 8.2 ± 1.3

1. (canceled)
 2. A composition comprising a therapeutic agent and anaqueous solution of a silk fibroin obtained from silkworm silk or spidersilk comprising biocompatible polymer, wherein the aqueous solution ofsilk fibroin comprising biocompatible polymer is free of solvents otherthan water.
 3. The composition of claim 2, wherein the aqueous solutionhas a shear rate from 24.3 to 1216 per second when measured using aCouette viscometer at room temperature.
 4. The composition of claim 2,wherein the aqueous solution has a conductivity of 19820 when measuredusing a Cole-Parmer conductivity meter at room temperature.
 5. Acomposition comprising (i) fibroin obtained from silkworm silk or spidersilk and (ii) a biocompatible polymer selected from polyethylene oxide,fibronectin, polyaspartic acid, polylysine, pectin, dextrans, andcombinations of two or more thereof.
 6. The composition of claim 5,further comprising an aqueous solution.
 7. The composition of claim 6,wherein the composition is free of solvents other than water.
 8. Thecomposition of claim 5, further comprising a therapeutic agent.
 9. Thecomposition of claim 8, wherein the therapeutic agent is selected fromthe group consisting of antiinfectives, chemotherapeutic agents,anti-rejection agents, analgesics and analgesic combinations,anti-inflammatory agents, hormones and growth factors.
 10. Thecomposition of claim 8, wherein the therapeutic agent is selected fromthe group consisting of antibiotics, antiviral agents, steroids, bonemorphogenic proteins, bone morphogenic-like proteins, epidermal growthfactor, fibroblast growth factor, platelet derived growth factor,insulin-like growth factor, transforming growth factors and vascularendothelial growth factor.
 11. The composition of claim 8, wherein thetherapeutic agent is selected from proteins, polysaccharides,glycoproteins and lipoproteins.
 12. The composition of claim 8, whereinthe therapeutic agent is present as a liquid or a finely divided solid.13. The composition of claim 8, further comprising one or moreadditives.
 14. The composition of claim 8, wherein the therapeutic agentis mixed with the biocompatible polymer.
 15. The composition of claim 8,wherein the therapeutic agent is coated on to the composition.
 16. Thecomposition of claim 8, wherein the amount of therapeutic agent is about0.001 percent to about 70 percent of the composition.
 17. Thecomposition of claim 8, wherein the therapeutic agent is released uponcontact with a body fluid.
 18. The composition of claim 8, wherein thebiocompatible polymer is polyethylene oxide.
 19. The composition ofclaim 5, wherein the composition is a fiber, a film, a foam or a mat.20. A method for delivering a therapeutic agent comprising administeringthe composition of claim 5 to a patient in need thereof.
 21. A methodfor repairing a wound comprising administering the composition of claim5 to a patient in need thereof.